This invention relates to a combination of immunotherapy and chemotherapy to promote tumor regression, particularly colorectal cancer and gastric carcinomas by treating a patient in need thereof with a combination of a humanized antibody that binds to A33 antigen and one or more chemotherapeutic agents. The combination of immunotherapy and chemotherapy is also useful for treatment of tumors that are resistant to one or more chemotherapeutic agents.
Colorectal cancer remains a major medical problem in the western world, with an estimated 130,000 new cases and 55,000 deaths in the United States in 1997 (Parker S. L., et al., Cancer statistics (1997). CA Cancer J Clin., 47:5-27 (1997)). In recent years, dietary and genetic factors have been identified as playing a role in determining an individual""s risk of developing the disease (Weisburger, xe2x80x9cCauses, relevant mechanisms, and prevention of large bowel cancerxe2x80x9d, Seminars in Oncology, 18:316-336 (1991) and Lynch et al. xe2x80x9cHereditary colorectal cancerxe2x80x9d, Seminars in Onocology; 18:337-366 (1991).
Adjuvant chemotherapy, radiation therapy, and immunotherapy have shown some usefulness in the treatment of primary and advanced colorectal carcinoma but additional agents and regimens are needed to consistently treat these diseases successfully (Petrelli et al., xe2x80x9cThe modulation of flourouracil with leucovorin in metastatic colorectal carcinoma: A prospective randomized phase III trialxe2x80x9d, J Clin Oncol, 7:1419-1426 (1989); Laurie et al., xe2x80x9cSurgical adjuvant therapy of large-bowel carcinoma: An evaluation of levamisole and the combination of levamisole and flourouracilxe2x80x9d, J Cin. Oncol.; 7:1447-1456 (1989); Byfield, et al., xe2x80x9cPharmacological requirements for obtaining sensitization of human cancer cells in vitro to combined 5-FU or ftorafur and X-raysxe2x80x9d, Int J Rad Oncol Biol Phys, 8:1923-1933 (1982); Mohiuddin and Marks, xe2x80x9cAdjuvant radiation therapy for colon and rectal cancerxe2x80x9d, Sem. Oncol.18:411-420 (1991); Krook, et al., xe2x80x9cEffective surgical adjuvant therapy for high-risk rectal carcinomaxe2x80x9d, N Engl. J. Med., 324:709-815 (1991) U.S. Pat. No. 5,851,526 and U.S. Pat. No. 5,958,412).
Chemotherapy alone has limitations in that the cancer cells often become resistant to a broad spectrum of structurally unrelated chemotherapeutic agents. Such resistance, termed xe2x80x9cmultidrug resistancexe2x80x9d (MDR), is not an uncommon problem in the treatment of patients with cancer and while significant efforts have been made to understand the mechanisms responsible for MDR, that understanding has not fulfilled the expectations for eradicating chemoresistant cancer cells.
Immunotherapy, alone or in combination with radiotherapy, has also been investigated as a method for inhibiting or eradicating cancer cells. In such treatments the immunoglobulin molecules are preferably specific for an antigen expressed on the cancer cells. In the case of colorectal cancers, radiolabelled antibodies specific for A33 antigen have shown promise for targeting colon cancer cells. The original mouse mAb A33 is an IgG2 a antibody that detects an epitope that is specific to colorectal cancer cells. Biopsy-based radioimmunolocalization studies quantitatively demonstrated high levels of antibody targeting to colorectal cancer.
A33 is a novel glycoprotein with a molecular weight of 43 kD in its monomeric form in Western blots under non-reducing conditions. Extensive immunohistochemical analysis of malignant and normal tissues has demonstrated that the antigen is homogeneously expressed by more than 95% of colon cancers and in the normal colon mucosa but not in other epithelial tissues. Immunohistochemical staining of the normal colon mucosa with serially diluted samples of mAb A33 suggests that A33 antigen expression is greatest at the top of the crypt and minimal at the base. A subset of gastric carcinomas also express the A33 antigen, while normal gastric mucosa are antigen-negative. The A33 antigen has been purified from human colon cancer cells, the protein sequence determined, the cDNA cloned, and the mouse homolog identified. See, U.S. Pat. No. 5,712,369.
The biodistribution and imaging characteristics of 131I-mAb A33 were studied in colon carcinoma patients with hepatic metastases. Control mAb TA99 studies showed that mAb A33 localization was antigen-specific, cancer:liver ratios were 2.3- to 45-fold higher for specific antibody as compared to non-specific antibodies. In metastatic lesions, the radioisotope is localized primarily in the viable periphery, but even the necrotic core concentrates mAb A33. External imaging shows isotope visualization in the bowel of some patients (Welt S. et al., xe2x80x9cQuantitative analysis of antibody localization in human metastatic colon cancerxe2x80x9d, A phase I study of monoclonal antibody A33, J Clin. Oncol.; 8:1894-1906(1990)).
mAb A33 localization is specific with regard to cancer blood pool (as determined by ratios of cancer:liver of injected 99Tc-HSA) and with regard to an isotype-matched control antibody. Autoradiographs of cancers and surrounding tissue of patients treated with radiolabeled antibody demonstrate that isotope accumulation in cancers corresponds to antibody binding specifically to the cancer cells while the surrounding stromal cells and vasculature do not concentrate the isotope.
A phase II study of 131I-A33 radioimmunotherapy (Welt S. et al., xe2x80x9cPhase I/II study of 131I-labeled monoclonal antibody A33 in patients with advanced colon cancerxe2x80x9d, J Clin Oncol, 12:1561-1571 (1994)) demonstrated 131I mAb-A33 had modest anticancer effects in heavily pre-treated patients who were no longer responding to chemotherapy. Of 23 patients treated, five had mixed responses: one patient displayed a disappearance of ascites and a drop in carcinoembryogenic antigen (CEA) levels from 4,200 ng/ml to 1,740 ng/ml, while other radiographic evidence of disease remained stable; in another patient with several pulmonary nodules, a single nodule disappeared while other evidence of disease remained stable; an additional patient with a nonmeasurable, pleura-based mass had a 30% decrease in CEA; and in another patient with progressive abdominal disease, several large lymph nodes of the neck disappeared. The fifth patient also showed a drop in CEA. These results show that murine 131I-mAb A33 has modest anti-cancer activity in heavily pretreated patients even after a single dose.
Five patients were treated with a second course of 131I-mAb A33 at 6-16 weeks; however, clearance of radiolabeled mAb was rapid, due to the human antimouse response (HAMA), and localization of isotope to the cancer sites was much less than after the first treatment, indicating that re-treatment is not practical.
An attractive feature of the A33 antigenic system for radioimmunotherapy is the in vitro evidence for rapid internalization of A33 antigen/antibody complexes into colon cancer cells (Daghighian et al., xe2x80x9cEnhancement of radiation dose to the nucleus by vesicular internalization of 125I-labeled A33 monoclonal antibodyxe2x80x9d, J. Nucl. Med.; 37:1052-1057 (1996)) and the ability of 125I-mAb A33-conjugates internalized into the cell to kill colon cancer cells in vitro and in a nuxe2x80x94nu mouse xenograft model. 125I-mAb A33 exerts its cytotoxic effects primarily through short-range Auger electrons, which are most effective if generated in close proximity ( less than 1-4 xcexcm) to the cell nucleus. Compared to xe2x80x9c3I-mAb A33, one of the expected benefits of 125I-mAb A33 is reduced bone marrow toxicity, and this expectation has been confirmed in the nuxe2x80x94nu mouse cancer model.
In a phase I/II study, twenty-one patients with advanced measurable or evaluable colon cancer who had failed at least one 5-fluorouracil (5-FU) based therapy but had not received prior radiotherapy, were treated with a single dose of 125I-labeled mAb A33 (Welt et al., xe2x80x9cPhase I/II study of Iodine 125-labeled monoclonal antibody A33 in patients with advanced colon cancerxe2x80x9d, J. Clin. Oncol., 14:1787-1797 (1996)). Of the 20 patients showing radiologic evidence of disease all displayed localization of 125I to sites of disease. Minor anti-cancer activity was also observed; levels of CEA returned to normal in one patient and decreased by 35% and 23% in two patients, respectively and one additional patient had a mixed response on CT. CEA is a marker associated with cancers, e.g., colon carcinoma and other metastatic cancers (Weinstein et al., xe2x80x9cAcute-phase proteins or tumour markers: The role of SAA, SAP, CRP and CEA as indicators of metastasis in a broad spectrum of neoplastic diseasesxe2x80x9d, Scand J Immunol; 19:193-8 (1984)). The use of the murine antibody specific for A33 was limited, due to the development of human antimouse antibody production (HAMA). Patients were on the study for only 6 weeks and were then given the option of chemotherapy.
Combination therapies, e.g., combinations of radiotherapy, chemotherapy and immunotherapy have been investigated in naive mice that had not been exposed to chemotherapy prior to the combination therapy (see e.g., Tschmelitsch et al., xe2x80x9cEnhanced Anticancer activity of combination radioimmunotherapy: 31I-Labeled Mononclonal Antibody A33 With Chemotherapy (Fluorouracil)xe2x80x9d, Cancer Res., 57:2181-2186 (1997)). Radioimmunotherapy with a radiolabeled mouse monoclonal antibody specific for the A33 antigen has been associated with a decrease in levels of serum CEA in patients who have failed conventional chemotherapy (Welt et al., J. Clin. Oncol., 14(6):1787-1797 (1996)). Some reports have suggested that the combination of chemotherapy and antibody-directed immune effector function enhances cancer cell lysis (Segerling et al., xe2x80x9cChemotherapeutic drugs increase killing of tumor cells by antibody and complementxe2x80x9d, Science, 188(4183):55-57 (1975) and Segerling et al., Enhancing effect by metabolic inhibitors on the killing of tumor cells by antibody and complementxe2x80x9d, Cancer Res., 35:3195-3203 (1975)). Agents with better anti-cancer activity and more effective treatment regimens are still needed if significant progress is to be made in the future.
Described herein is a method for promoting tumor regression by administering one or more chemotherapeutic agents and an antibody specific for the A33 antigen to patients in need thereof. The methods are useful for treating patients whose tumors are chemoresistant to one or more chemotherapeutic agents. The methods are suitable for treating patients having solid tumors and/or tumors that have metastasized. The methods described herein are suitable for use with patients whose tumors are metastatic and chemoresistant. Patients having chemoresitant cancers responded unexpectedly well to the combination of immunotherapy and chemotherapy as compared to the chemotherapy or immunotherapy alone. The methods promoted cancer regression even where the antibody was a non-radiolabelled antibody. In addition, these positive results in patients with chemoresistant tumors suggest that the treatment regimen described herein would also be appropriate for the treatment of patients whose tumors had not become chemoresistant.
This invention relates to methods for promoting regression of cancer cells, particularly those expressing A33, with a combination of chemotherapeutic agents and immunotherapy. Previously, multidrug combinations, e.g., MOF-Strep (Methyl CCNU, fluorouracil, vincristine and streptozocin) showed significant responses in two initial trials (32-34% overall major response rated) but, in subsequent larger studies, the response rate was lower, resulting in a loss of interest in this combination as an effective cancer treatment. (Kemeny et al., xe2x80x9cTherapy for metastatic colorectal carcinoma with a combination of methyl-CCNU, 5-fluorouracil, vincristine and streptozocinxe2x80x9d, Cancer 45:876-881(1980); Kemeny et al., xe2x80x9cMetastatic colorectal carcinoma: A prospective randomized trial of methyl-CCNU, 5-flourouracil (5-FU) and vincristine (MOF) versus MOF plus streptozocin (MOF-Strep)xe2x80x9d, Cancer, 51:20-24 (1983)). Recently, there has been renewed interest in combinations of BCNU (carmustine) and streptozocin due to their synergistic activities (see, e.g., Wilson et al., xe2x80x9cModulation of O6-alkylguanine alkyltransferase-directed DNA repair in metastatic colon cancersxe2x80x9d, J. Clin Oncol 1995; 13:2301-2308 (1995) and Preuss et al., xe2x80x9cProtective effect of O6-methylguanine-DNA methyltransferase (MGMT) on the cytotoxic and recombinogenic activity of different antineoplastic drugs.xe2x80x9d Int J Cancer, 65:506-512 (1996)). In addition, immune-mediated mechanisms such as ADCC have been shown to be potentiated with cytotoxic reagents (Pfeifer and Bosmann, xe2x80x9cModulation of anticancer antibody-dependent cellular cytotoxicity and natural killer activity by Adriamycin and daunorubicinxe2x80x9d, Agents Action, 12:635-644 (1982), and Schlager and Ohanian, xe2x80x9cRole of membrane lipids in the immunological killing of cancer cells: I. Target cell lipidsxe2x80x9d, Lipids, 18:475-482(1983)).
The methods of this invention are particularly efficacious for patients having cancer cells that display resistance to one or more chemotherapeutic agents, e.g., the growth rate of the resistant cancer cells is not retarded, new cancer foci continue to emerge, and/or the cancer foci do not display significant reduction in size in response to chemotherapeutic treatments. The chemoresistant cancers may be resistant to the same chemotherapeutic agent that is used in combination with the A33-specific antibody. Co-assigned U.S. Pat. Nos. 5,851,526 and 5,958,412, incorporated herein by reference, disclose methods for treating colon cancer using cancer-specific antibodies conjugated with a radioisotope or an anti-cancer drug. It is disclosed herein that a higher than expected proportion of patients responded favorably to the regimen of chemotherapeutic agents in combination with A33 specific antibodies. Thus far 3/12 patients responded favorably in sharp contrast to treatments with only chemotherapeutic agents where only 10% of patients with chemoresistant tumors responded to the treatment. The patients described herein responded well even though the antibody was not labeled with a radioisotope or other anticancer label. This is the first demonstration in humans of an enhancement of antitumor effects in solid tumors when immune mediated killing of cancer cells directed by antibodies is combined with chemotherapy.
Described herein is the a method for promoting regression of cancer cells expressing A33 antigen by treating a patient having such cancer cells with a combination of immunotherapy and chemotherapy, preferably an antibody specific for A33 antigen and one or more chemotherapeutic agents. The A33-specific antibody(s) and one or more chemotherapeutic agents are administered in sufficient amounts to promote regression of the cancer. Sufficient amounts slow the growth rate of cancer cells, reduce the appearance of new cancer foci, reduce the size of tumors and/or reduce the levels of serum CEA. The chemotherapeutic agents may be administered prior to, concurrently with, or after A33 specific antibody is administered to the patient. Preferably the A33 specific antibody and chemotherapeutic agents are administered concurrently. More preferably the A33 specific antibody is used in combination with one or more chemotherapeutic agents selected from the group consisting of oxaliplatin, irinotecan, topotecan, carmustine, vincristine, fluorouracil, leucovorin and streptozocin. Even more preferably the chemotherapeutic agent is selected from the group consisting of carmustine, vincristine, fluorouracil, leucovorin and streptozocin. Most preferably the A33 specific antibody is administered in conjunction with BOF-Strep (carmustine, vincristine, fluorouracil, and steptozocin).
The A33 specific antibody may be chimeric, multimeric, hetreomeric or single chain form of the antibody. For example the antibody may be a diabody, a triabody or a tetrabody. Preferably the A33 specific antibody is a humanized antibody.
The methods described herein comprise administering to a patient in need thereof a combination of chemotherapeutic agents and immunoglobulins specific of an antigen expressed on cancer cells. Preferably the antigen is an A33 antigen and preferably the immunoglobulin molecule is a humanized immunoglobulin molecule. More preferably a humanized antibody is one with a strong effector cell-mediated cytotoxicity. Strong effector cell mediated cytotoxicity is measured using routine methods well known in the art such as e.g, a chromium release assay. Preferably the chemotherapeutic agents and immunoglobulins are administered to the patient concurrently. The combination of chemotherapy, particularly BOF-strep (carmustine, fluorouracil, vincristine and streptozocin) and immunotherapy applied to subjects having chemoresistant cancer cells, i.e., cancer cells that had become resistant to one or more chemotherapeutic agents, promote unexpectedly higher cancer cell regression than chemotherapy or immunotherapy alone. Patients with chemoresistant cancer cells were not expected to respond to the combination of immunotherapy and chemotherapy any better than patients with chemoresitant cancers who were treated with only immunotherapy or chemotherapy.
Those of skill in the art who routinely treat patients with colorectal cancer are aware that there are many different regimens for the treatment of such cancers and the application of those regimens to particular patients will depend on the consideration of a variety of factors, e.g., the stage of the cancer, the extent of the spread of the cancer cells, e.g. have they metastasized, and the physical attributes of the patient. Those of skill in the art routinely adjust the parameters of a particular treatment, e.g., dose, duration, administration route and administered form, for particular patients and those parameters are adjusted without undue experimentation by one of ordinary skill in the art. Many chemotherapeutic protocols, the criteria for their modification and the factors which contraindicate their application are set forth in, e.g., Table 1, and reviews in Seminars in Oncology, 26(5) and 26(6) (1999).
The results presented herein demonstrate that the addition of A33 specific antibodies to a chemotherapeutic regimen effectively promotes regression of cancer cells that had developed resistance to chemotherapeutic agents. The regression is manifested by slowing the growth rate, reducing the incidence of new cancer foci emerging, and/or reducing the size of the cancer foci. Regression of the cancer can also be measured by assaying the CEA levels within the treated patients. A reduction in CEA levels is indicative of a regression of the cancer mass. CEA levels may be measured using any method known in the art.
The methods described herein combine administering an antibody specific for the A33 antigen and at least one, and preferably a combination, of chemotherapeutic agents. Preferably the chemotherapeutic agents are oxaliplatin, irinotecan, topotecan, leucovorin, carmustine, vincristine, fluorouracil and/or streptozocin. These chemotherapeutic agents are commercially available to those of skill in the art. More preferably the A33-specific antibody is combined with one or more chemotherapeutic agents selected from the group consisting of carmustine, vincristine, fluorouracil or streptozocin. Most preferably the A33 specific antibody is administered in combination with BOF-Strep (carmustine, vincristine, fluorouracil or streptozocin).
Preferably the A33 specific antibody is a monoclonal antibody. Monoclonal antibodies are obtainable via many methods well known in the art. For example, the Kohler-Millstein method, is one such well-known method comprising immunizing an experimental animal, preferably a mouse, with the appropriate protein, isolating antibody-producing B cells or spleen cells from the immunized experimental animal and subsequently fusing the antibody-producing cells with a suitable leukemia cell to produce hybridomas (Kohler and Millstein, xe2x80x9cDerivation of specific antibody-producing tissue culture and cancer lines by cell fusionxe2x80x9d Eur. J Immunol. 6:511-519 (1976); Kohler-Millstein et al., Methods Enz. 73:1 (1981)). Monoclonal antibodies may also be generated and isolated from phage display libraries. The construction and screening of phage display libraries are well known in the art, see, e.g., Hoogenboom, xe2x80x9cDesigning and optimizing library selection strategies for generating high affinity antibodiesxe2x80x9d Trends Biotechnol., 15:62-70 (1997); Hoogenboom, et al. xe2x80x9cAntibody phage display technology and its applicationsxe2x80x9d, Immunotechnology 4:1-20 (1998); McGregor, xe2x80x9cSelection of proteins and peptides from libraries displayed o filamentous bacteriophagexe2x80x9d, Mol. Biotechnol, 6:155-62 (1996); Bird et al., xe2x80x9cSingle-chain antigen binding proteinsxe2x80x9d, Science, 242:423-426 (1988); Perisic et al., Structure 2:1217-1226 (1994); Pei et al., PNAS, 94:9637-9642 (1997); Hollinger et al., Protein Engineering 9:299-305 (1996); Millstein and Cuello, Nature 305, 537-539 (1983); Yamanaka et al. (1996). xe2x80x9cChicken monoclonal antibody isolated by a phage display system.xe2x80x9d J Immunol., 175(3): 1156-1162 (1996) all incorporated herein by reference.) More preferably the monoclonal antibody is a humanized monoclonal antibody.
Humanized antibodies may be chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fabxe2x80x2, F(abxe2x80x2)2 or other antigen-binding sub-sequences of antibodies) containing a minimal amount of sequence derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins in which complementarity-determining regions (CDR) are replaced by CDRs of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity and/or affinity. Methods for the production of humanized antibodies are also known in the art. See e.g., Cabilly, et al., U.S. Pat. No. 4,816,567; Mage and Lamoyi, in Monoclonal Antibody Production Techniques and Applications, pp. 79-97 (Marcel Dekker, Inc., New York, 1987); Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992). Preferably the humanized A33 antibody is a fully humanized CDR-grafted A33 IgG1 antibody (huA33).
The A33-specific antibody may be a chimeric, multivalent, multimeric, or heteromeric form of the antibody. For example, the antibody may be a diabody, a single chain Fv, a triabody or a tetrabody. Those of skill in the art are familiar with methods for the production of such antibody forms, see e.g., Perisic et al., Structure 2:1217-1226 (1994); Pei et al., PNAS, 94:9637-9642 (1997); Hollinger et al., Protein Engineering 9:299-305 (1996); Millstein and Cuello, Nature 305, 537-539 (1983) incorporated herein by reference.
An A33 specific antibody may be administered with chemotherapeutic agents and with another antibody conjugate. The additional antibody may be conjugated with a radioisotope or another chemotherapeutic or cytotoxic agent. The radioisotope may be for example, 125I, 131I, 99Tc, 90Y and 111In. The A33-specific antibody may also be conjugated to a radioisotope e.g., 125I, 131I, 99Tc, 90Y, 111In and other xcex1, xcex2 and Auger emitters, or a chemotherapeutic or cytotoxic agent. Those of skill in the art will appreciate that there are many suitable methods for the conjugation of an antibody with a radioisotope, e.g., U.S. Pat. Nos. 5,160,723 and 5,851,526, incorporated herein by reference. The antibody may be conjugated with a chemotherapeutic agent, such as those recited supra, or a cytotoxic agent. For example, huA33 can be conjugated with QFA, which is an antifolate, or with calicheamicin, which is an anti-cancer antibiotic that cleaves double-stranded DNA of cancer cells. Both QFA and calicheamicin have intracellular sites of action and do not readily cross the plasma membrane. As such, they have weak cytotoxic effects when added to cell cultures. Cellular uptake of these agents through huA33-mediated internalization greatly enhances their cytotoxic effects in vitro. In vivo xenograft studies show that cancer inhibition with limited normal tissue damage can be obtained with both huA33-QFA and huA33-calicheamicin conjugates. Other conjugation partners can also be conjugated to the antibodies of the invention e.g., enzymes, prodrugs, and cytotoxic agents e.g., BCNU, mercaptopurine, methotrexate or adriamycin. Any method known in the art for preparing antibody conjugates may be used to generate the conjugates of this invention.
The chemotherapeutic agents may be administered prior to, concurrently with, or after the A33 specific antibody has been administered to the patient. Preferably the A33 specific antibody is administered to a patient repeatedly, preferably in one week intervals. More preferably the A33 specific antibody is administered to the patient once a week for at least about 10 weeks, preferably at least about 14 weeks. Each administration of the A33 specific antibody is preferably a pharmaceutically effective amount, so as to reduce the effects of colon cancer, preferably about 2 to about 100 mg/m2. Preferably the patient in need thereof is pretreated with a regimen of A33-specific antibody without a chemotherapeutic agent before the chemotherapeutic agents are added to the regimen for concurrent treatment with both the antibody and the chemotherapeutic agent(s). Preferably A33 specific antibody is administered to the patient for several weeks, preferably about 4 to 6 weeks, more preferably about 5 weeks, before the chemotherapeutic agents are added to the regimen.
The chemotherapeutic agents, oxaliplatin, irinotecan, topotecan, leucovorin, carmustine, vincristine, fluorouracil and/or streptozocin, may be administered to the patient sequentially or concurrently or in combinations that are used routinely in the art. For example, in one embodiment, carmustine, vincristine, fluorouracil or streptozotocin (BOF-Strep) are administered to the patient in a regimen that is similar to that described by Kemeny et al., xe2x80x9cMetastatic colorectal carcinoma: A prospective randomized trial of methyl-CCNU, 5-fluorouracil (5-FU) and vincristine (MOD) versus MOF plus streptozocin (MOF-Strep)xe2x80x9d, Cancer, 51:20-24 (1983) incorporated herein by reference). BOF-Strep may be administered to the patient then, preferably, about one day later and then once a day for several days, preferably about 4 days, carmustine and fluorouracil but not vincristine or streptozocin are administered to the patient. Thereafter streptozocin, but not carmustine, fluorouracil or vincristine, is administered to the patient once a week for about 4-6 weeks, preferably about 4 weeks. This administration of streptozocin is followed by administration of fluorouracil, vincristine, and streptozocin, preferably a single dose of these three agents is administered to the patient. After the administration of the fluorouracil, vincristine, and streptozocin, fluorouracil, but not carmustine, vincristine or streptozocin are administered to the patient once a day for about 3 to 6 consecutive days, preferably about 4 days. Thereafter, streptozocin, but not carmustine, fluorouracil or vincristine is administered to the patient once a week for about 3 to 6 weeks, preferably about 4 weeks. During the administration of the chemotherapeutic agents the patient is also receiving the A33 specific antibody about once a week. This regimen of chemotherapeutic agents and A33 specific antibody may be repeated as often as desired. Preferably carmustine is administered at a dose of about 20 mg/m2 to about 40 mg/m2, more preferably about 30 mg/m2. Preferably vincristine is administered at a dose of about 0.5 mg/m2 to about 2 mg/m2, more preferably about 1.0 mg/m2. Preferably fluorouracil is administered at a dose of about 200 mg/m2 to about 400 mg/m2, more preferably about 300 mg/m2. Preferably, streptozocin is administered at a dose of about 250 mg/m2 to about 750 mg/m2, more preferably about 500 mg/m2. Those of skill in the art can easily determine suitable dosages of each chemotherapeutic agents depending on the characteristics of the individual patient, e.g., the size of the patient and on the response of the patient to the agents. For example, Table 1 demonstrates art-accepted suggestions for the quantity of the agents.
The A33 specific antibody may be a humanized antibody. Methods for humanizing antibodies are well known in the art, see e.g., Daugherty et al., Nucl. Acids Res., 19:2471-2476 (1991), King et al, Cancer Res., 54:6176-6185 (1994), and U.S. Pat. No. 5,821,526, incorporated herein by reference. Preferably the humanized antibody is a fully humanized monoclonal antibody. In one embodiment of this invention a fully humanized CDR-grafted A33 IgG1 (huA33) was used. This humanized IgG1 version of mouse A33 antibody activates immune effector function and in phase I-II trials, huA33 alone was found to have modest anti-cancer activity. HuA33 is equivalent to murine mAb A33 in competitive binding assays and in localization studies in a nuxe2x80x94nu mouse model (Tschmelitsch et al., Cancer Res., 57:2181-2186 (1997)). Radio-iodinated huA33 retains immunoreactivity and repeated administration of huA33 to cynomolgus monkeys for up to one year demonstrates that the antibody is not immunogenic in primates; however, unlike the original mouse IgG2a antibody, huA33 has strong effector cell-mediated cytotoxicity, especially against A33-expressing cancer cell lines which retain  greater than 400,000 antibody molecules on their cell surface after the bulk of the bound huA33 is internalized.